After we had finished the experiments, assessment of how well we did was made.

+

+

; Goal 1

+

: Not only the DNA shell structure is confirmed using AFM but also the captured state was analysed geometrically. Therefore, we believe that this goal is accomplished.

+

; Goal 2.

+

: As for the detection, we were able to confirm that by using more than one pair of fluorescent and quencher molecules, we were able to get a higher signal of the capturing. We also confirmed in the experiment of using DNA Shell to protect the HRP-Streptavidin that the activity of streptavidin could be retained even if it was in a captured state. Finally, the experiments showed that the attachment of DNA shell to the microfluidics device was also achieved.

+

+

==Improvement==

+

To improve and give a more useful result to verify our idea, these experiments below should be conducted.

+

+

; Finding the length of each fluorescent and quencher molecules where self-absorption does not occur

+

: When we added 12 equiv. pairs of fluorescent and quencher molecules, there was no difference in the change in fluorescence. This is thought to be caused by a phenomena called self-absorption, where the emitted fluorescence is captured by another fluorescent molecule. This can be prevented by enlarging the distance between the fluorescent molecules. Finding the length this phenomena will not be troubling would be a method to make this more device useful.

+

; Protection experiment using new Trypsin reagent and also with different groups of proteases

+

: Our experiment with Trypsin seemed not to work because of the failure in Trypsin activity. Therefore, we would like to do the experiment again with a new Trypsin. Also, it would be better to try with different proteases.

+

+

=Future Works=

+

From the results of our experiments, we confirmed that the proposed ideas were realized. To give ideas on a more broader span of ideas, we also thought of places this new nano-device could work in handy.

+

+

==Finding New Aptamers for New and Different Substrates==

We made DNA Shell like below (Fig.1), and additional DNA that specifically binds to a certain substance can be constructed on the Shell. By changing the arrangement of the DNA, you can capture whatever substance you like.

We made DNA Shell like below (Fig.1), and additional DNA that specifically binds to a certain substance can be constructed on the Shell. By changing the arrangement of the DNA, you can capture whatever substance you like.

We thought of capturing thrombin. We could not experiment because of the shortage of time, but we made arrangements of the DNA that bind to thrombin, the thrombin aptamer. A part of the thrombin aptamer forms G-quartet structure to which thrombin binds(Fig. 3) (W Yoshida, K Sode, K Ikebukuro, ''Anal. Chem.'', 2006, 78 (10), pp 3297–3299).

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+

<center>

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<gallery widths="400" heights="300" perrow="1">

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Image:Biomod-2012-UTokyo-UT-Hongo-FutureWorkthrombinaptamer.jpg|Fig.3

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</gallery>

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</center>

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+

Putting a part of thrombin aptamer on the center plane and other non-fixed plane of Shell (Fig. 4), Shell would close when thrombin comes near(Fig. 5).

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<gallery widths="330" heights="200" perrow="2">

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Image:Biomod-2012-UTokyo-UT-Hongo-FutureWorkShell.jpg|Fig.4

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Image:Biomod-2012-UTokyo-UT-Hongo-FutureWorkcapturethrombin.jpg|Fig.5

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</gallery>

-

We thought of capturing ATP (Fig.3) or thrombin. We could not experiment because of the shortage of time, but we made arrangements of the DNA that bind to ATP or thrombin.

Thrombin solidifies the blood, so the Shell that specifically binds to thrombin can prevent a blood clot.

Thrombin solidifies the blood, so the Shell that specifically binds to thrombin can prevent a blood clot.

Like these, you can change the arrangement of the DNA on the Shell, and capture many kinds of substances.

Like these, you can change the arrangement of the DNA on the Shell, and capture many kinds of substances.

It makes DNA Shell a more useful device.

It makes DNA Shell a more useful device.

-

-

<br style="clear: both;">

=DNA Shell with Functionality=

=DNA Shell with Functionality=

By putting DNA Shell and an enzyme together, you can use the Shell as a more functional device.

By putting DNA Shell and an enzyme together, you can use the Shell as a more functional device.

+

As example, we experimented with tetramethylbenzidine(TMB), horseradish peroxidase(HRP), and trypsin.

As example, we experimented with tetramethylbenzidine(TMB), horseradish peroxidase(HRP), and trypsin.

-

Like that experiment, by capturing the substrate, you can use the Shell as an inhibitor for enzyme (Fig.4).

When attaching a biotin to the non-binding site of the DNA that is released in the strand displacement reaction, without the input DNA, streptavidin cannot combine with biotins because of the steric hindrance. However, once the DNA is input, the combination between streptavidin and biotins can be formed (Fig. 8).

The combination is formed only when two, three, or four biotin monomers are binding to one streptavidin. Two strand displacement reaction devices can be constructed on both sides of the Shell (total four devices) (Fig. 9).

Each device catches the different input DNA. When the more than two biotins on the different Shell sides are gathering, the Shell will be closed (i.e., biotins on the A and B, or C and D gathering, the Shell will not be closed). So you can use four inputs.

<br style="clear: both;">

<br style="clear: both;">

-

=A Device more than Shell and Enzyme=

-

When you combine the features of the above 1. ~3., you can get the more advanced DNA Shell.

-

For example, in the case of using the Shell with the logical circuit, the Shell captures a substrate only when all the four kinds of input DNA are exist.

Progress of Our Research

After we had finished the experiments, assessment of how well we did was made.

Goal 1

Not only the DNA shell structure is confirmed using AFM but also the captured state was analysed geometrically. Therefore, we believe that this goal is accomplished.

Goal 2.

As for the detection, we were able to confirm that by using more than one pair of fluorescent and quencher molecules, we were able to get a higher signal of the capturing. We also confirmed in the experiment of using DNA Shell to protect the HRP-Streptavidin that the activity of streptavidin could be retained even if it was in a captured state. Finally, the experiments showed that the attachment of DNA shell to the microfluidics device was also achieved.

Improvement

To improve and give a more useful result to verify our idea, these experiments below should be conducted.

Finding the length of each fluorescent and quencher molecules where self-absorption does not occur

When we added 12 equiv. pairs of fluorescent and quencher molecules, there was no difference in the change in fluorescence. This is thought to be caused by a phenomena called self-absorption, where the emitted fluorescence is captured by another fluorescent molecule. This can be prevented by enlarging the distance between the fluorescent molecules. Finding the length this phenomena will not be troubling would be a method to make this more device useful.

Protection experiment using new Trypsin reagent and also with different groups of proteases

Our experiment with Trypsin seemed not to work because of the failure in Trypsin activity. Therefore, we would like to do the experiment again with a new Trypsin. Also, it would be better to try with different proteases.

Future Works

From the results of our experiments, we confirmed that the proposed ideas were realized. To give ideas on a more broader span of ideas, we also thought of places this new nano-device could work in handy.

Finding New Aptamers for New and Different Substrates

We made DNA Shell like below (Fig.1), and additional DNA that specifically binds to a certain substance can be constructed on the Shell. By changing the arrangement of the DNA, you can capture whatever substance you like.
As example, we experimented with biotin and streptavidin (Fig.2).

Fig.1

Fig.2

We thought of capturing thrombin. We could not experiment because of the shortage of time, but we made arrangements of the DNA that bind to thrombin, the thrombin aptamer. A part of the thrombin aptamer forms G-quartet structure to which thrombin binds(Fig. 3) (W Yoshida, K Sode, K Ikebukuro, Anal. Chem., 2006, 78 (10), pp 3297–3299).

Fig.3

Putting a part of thrombin aptamer on the center plane and other non-fixed plane of Shell (Fig. 4), Shell would close when thrombin comes near(Fig. 5).

Fig.4

Fig.5

Thrombin solidifies the blood, so the Shell that specifically binds to thrombin can prevent a blood clot.
Like these, you can change the arrangement of the DNA on the Shell, and capture many kinds of substances.
It makes DNA Shell a more useful device.

DNA Shell with Functionality

By putting DNA Shell and an enzyme together, you can use the Shell as a more functional device.

As example, we experimented with tetramethylbenzidine(TMB), horseradish peroxidase(HRP), and trypsin.

Like that experiment, by capturing the substrate, you can use the Shell as an inhibitor for enzyme (Fig.6).

Fig.6

Shell with a Logical Circuit

To get more advanced DNA Shell with a logical circuit, you can make the toehold medicated strand displacement reaction device on the Shell.
In the strand displacement reaction, when a DNA is input, another DNA is released.

Fig.7

Design of the Logical Gates on DNA Shell

When attaching a biotin to the non-binding site of the DNA that is released in the strand displacement reaction, without the input DNA, streptavidin cannot combine with biotins because of the steric hindrance. However, once the DNA is input, the combination between streptavidin and biotins can be formed (Fig. 8).

Fig.8

The combination is formed only when two, three, or four biotin monomers are binding to one streptavidin. Two strand displacement reaction devices can be constructed on both sides of the Shell (total four devices) (Fig. 9).

Fig.9

Each device catches the different input DNA. When the more than two biotins on the different Shell sides are gathering, the Shell will be closed (i.e., biotins on the A and B, or C and D gathering, the Shell will not be closed). So you can use four inputs.